Formation of Nuclear Bodies in Cells Overexpressing the Nuclear Pore Protein POM121

Söderqvist, Henrik; Jiang, Wei‐Qin; Ringertz, Nils R.; Hallberg, Einar

doi:10.1006/excr.1996.0158

Cited by 21 publications

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“…COS-1 cells (African green monkey kidney cells) were cultured at 37°C on 13-mm glass coverslips as described (Soderqvist et al, 1996) ; 75 % confluent monolayer cultures were transfected using the polycationic reagent N- [ 1 -(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsuphate (DOTAP liposomal transfection regent, Boehringer Mannheim) according to the manufacturer's manual ; 5 h after transfection in serum-free medium the cell cultures were allowed to incubate for an additional 19 h or 43 h at 37OC in normal culture medium. The human neuroblastoma SH-SY5Y cells were grown in modified Eagle medium containing 2 mM glutamine, 1 % non-essential amino acids, 15 % heat-inactivated fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin.…”

Section: Methodsmentioning

confidence: 99%

“…POM121 and do not crossreact with the endogenous COS cell homologue (Soderqvist et al, 1996). Transfected COS-1 cells grown on 13-mm coverslips were prepared for immunofluorescence as described (Soderqvist et al, 1996).…”

Section: '-Aaaaagcttgccgcgatggtgtttggctttggagtg)mentioning

confidence: 99%

“…Transfected COS-1 cells grown on 13-mm coverslips were prepared for immunofluorescence as described (Soderqvist et al, 1996). Affinity purified rabbit P9107 antibodies (see above), mouse monoclonal mAb414 antibodies (Davis and Blobel, 1986) or rabbit polyclonal antibodies raised against a synthetic peptide corresponding to amino acids 556-573 of the endoplasmic reticulum protein calnexin (Wada et al, 1991) were used as primary antibodies.…”

Section: '-Aaaaagcttgccgcgatggtgtttggctttggagtg)mentioning

confidence: 99%

“…In a previous study we showed that rat POM121, or its Cterminal portion alone, displayed a punctate distribution in the nuclear envelope of COS-1 cells when overexpressed at low levels and formed intranuclear bodies when overexpressed at high levels (Soderqvist et al, 1996). In that study overexpressed rat POM121 was monitored by affinity purified species-specific antibodies raised against a synthetic peptide, P9107 (corresponding to amino acids 485-496 of POM121; Hallberg et al, 1993).…”

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Intracellular Distribution of an Integral Nuclear Pore Membrane Protein Fused to Green Fluorescent Protein — Localization of a Targeting Domain

Söderqvist

Imreh

Kihlmark

et al. 1997

European Journal of Biochemistry

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The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticuludnuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121.Keywords : nuclear pore ; endoplasmic reticulum ; protein trafficking ; green fluorescent protein.The eukaryotic cell nucleus is surrounded by the nuclear envelope, a double lipid membrane that separates the cytoplasm from the nucleoplasm (for reviews see Gerace and Burke, 1988;Miller et al., 1991). At numerous circumscribed points, referred to as the pore membrane domains of the nuclear envelope, the outer and inner nuclear membranes of the nuclear envelope are fused together to form nuclear pores. The nuclear pore complexes (NPCs) are multiprotein structures embedded in the nuclear pores and constitute the exclusive sites of nucleokytoplasmic transport (Pant6 and Aebi, 1996). The pore membrane proteins, i.e. integral membrane proteins of the nuclear pore complex, are believed to function in pore formation and assembly of the NPC (Hallberg et al., 1993;Wozniak et al., 1989Wozniak et al., , 1994. The cDNA of the 121-kDa pore membrane protein (POM121) was cloned and sequenced and its sequence was deduced (Hallberg et al., 1993). The protein has a short luminally exposed N-terminal tail, a single transmembrane domain and a large C-terminal portion adjoining the NPC (Soderqvist and Hallberg, 1994). The C-terminal third of POM121 contains several pentapeptide XFXFG repeats in common with a subfamily of peripheral nuclear pore complex proteins and are believed to

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Section: Methodsmentioning

confidence: 99%

Section: '-Aaaaagcttgccgcgatggtgtttggctttggagtg)mentioning

confidence: 99%

Section: '-Aaaaagcttgccgcgatggtgtttggctttggagtg)mentioning

confidence: 99%

mentioning

confidence: 99%

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Intracellular Distribution of an Integral Nuclear Pore Membrane Protein Fused to Green Fluorescent Protein — Localization of a Targeting Domain

Söderqvist

Imreh

Kihlmark

et al. 1997

European Journal of Biochemistry

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“…A marked difference between these two proteins is the size of their lumenal and pore-side domains. Whereas most of the mass of gp210 is positioned in the lumen of the NE (5,8), the majority of Pom121p is on the pore side of the membrane, including a region containing repetitive sequences of the type FXFG also found in a subset of nucleoporins (6,9).…”

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Topology and Functional Domains of the Yeast Pore Membrane Protein Pom152p

Tcheperegine

Marelli

Wozniak³

1999

Journal of Biological Chemistry

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Integral membrane proteins associated with the nuclear pore complex (NPC) are likely to play an important role in the biogenesis of this structure. Here we have examined the functional roles of domains of the yeast pore membrane protein Pom152p in establishing its topology and its interactions with other NPC proteins. The topology of Pom152p was evaluated by alkaline extraction, protease protection, and endoglycosidase H sensitivity assays. The results of these experiments suggest that Pom152p contains a single transmembrane segment with its N terminus (amino acid residues 1-175) extending into the nuclear pore and its C terminus (amino acid residues 196 -1337) positioned in the lumen of the nuclear envelope. The functional role of these different domains was investigated in mutants that are dependent on Pom152p for viability. The requirement for Pom152p in strains containing mutations allelic to the NPC protein genes NIC96 and NUP59 could be alleviated by Pom152p's N terminus, independent of its integration into the membrane. However, complementation of a mutation in NUP170 required both the N terminus and the transmembrane segment. Furthermore, mutations in NUP188 were rescued only by full-length Pom152p, suggesting that the lumenal structures play an important role in the function of pore-side NPC structures.Bidirectional transport between the nucleus and the cytoplasm is mediated by nuclear pore complexes (NPCs) 1 (1, 2). These massive structures extend across the nuclear envelope (NE) and are bound to the pore membrane domain (see Refs.

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Assays using digital fluorescence: 1985-1998

Kricka¹,

Stanley²

1999

Luminescence

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Formation of Nuclear Bodies in Cells Overexpressing the Nuclear Pore Protein POM121

Cited by 21 publications

References 0 publications

Intracellular Distribution of an Integral Nuclear Pore Membrane Protein Fused to Green Fluorescent Protein — Localization of a Targeting Domain

Intracellular Distribution of an Integral Nuclear Pore Membrane Protein Fused to Green Fluorescent Protein — Localization of a Targeting Domain

Topology and Functional Domains of the Yeast Pore Membrane Protein Pom152p

Assays using digital fluorescence: 1985-1998

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